A Review of Seeding Methods

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A Review of Seeding Methods

• Allan DArcy, Aengus Mac Sweeney, Alex Haber
• RAMC, 2005

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Optimisation of crystal quality

• Obvious First Steps
• Vary initial conditions
• Protein concentration, precipitating agent,
  salt, pH, temperature
• Additives
• Detergent, glycerol, ethylene glycol, organic
  solvents etc.
• Setup type
• Microbatch, hanging drop, sitting drop, oil in
  reservoir
• Seeding
• Microseeding, macroseeding

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Nucleation and seeding
50 of proteins require seeding to obtain good
crystals
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Nucleation and seeding
Nucleation Different types of seeding Screening
with seeding
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Nucleation
The formation of a core that allows further
crystal growth
The nucleation event is a parameter that can be
difficult to control. A sufficiently high level
of saturation is often not reached and the
critical nucleation event cannot occur. Local
variations in protein and precipitant
concentration favour nucleation.
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Nucleation and crystal growth
Many drops remain clear during screening
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A Real Phase Diagram
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What affects nucleation?
Sample concentration Sample preparation
(filtering, storage) Method used VD, MB,
FID Mixing Surfaces
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Mixing drops on set up
Unmixed drops sample a greater range of protein
and precipitant concentrations nucleation is
more likely
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Uses for seeding
Reproducing difficult crystallizations Obtaining
favoured crystal form Cocrystallizing with
inhibitors Improve screening success Screening
for crystal growth conditions
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Different Seeding Methods (I)
Macro
With fresh crystals the crystal washing step is
often unnecessary
More focused X-ray sources reduce need for
macroseeding
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Different Seeding Methods (II)
Micro
Streak seeding with needle or hair
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Seed anything that might be microcrystalline
(Terese Bergfors)
FabD spherulites
(Spherulites are crystalline test diffraction)
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Different Seeding Methods (III)
Microdilution
Seed Beads
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Microseeding
Dilution series of stock made with seed bead
Concentrated seed stock
15 diluted seed stock
125 diluted seed stock
Same protein streak and micro (bead) seeded
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Microseed matrix screening
Microseed matrix screening to improve crystals of
yeast cytosine deaminase. Ireton, G.C.
Stoddard, B.L. Acta Crystallogr D (2004), 60(3),
601-5.
A crystallization strategy termed microseed
matrix screening is described where the optimal
conditions for nucleation versus extended lattice
growth are not compatible.
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Influencing nucleation at the start
Problems We need seeds The seeds must be stable
in the initial conditions
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The nucleation problem

• McPherson Schlichta 1987 Crushed mineral
  materials
• Punzi et al. 1991 Polyvinylidene difluoride
• Chayen et al. 2001 Porous silicon
• Fermani et al. 2002 Modified mica
• Haushalter McPherson 2002 Nanoengineered
  Surfaces
• Rong et al. 2004 Porous glass

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Influencing nucleation using growth surfaces
Built-in Heterogeneous Seeding
Parallel Synthesis Technologies, Inc.
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Natural heterogeneous seeding?
Serine protease
Viral serine protease
Cysteine protease
Elastase
Cysteine protease
Tryptase
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Crystals of chicken triose phosphate isomerase
(the first TIM barrel) taken in 1970
Courtesy of David Banner
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Horse hair is used for streak seeding and has a
complex surface structure
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Nucleation on horse hair
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Using the sonicated stock
Control no hair added
Diluted hair added
Concentrated hair added
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Crystals growing on crushed horse hair
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Testing on different proteins
Protein Control Seed dilution
(0 seeds) 15 125 Trypsin 0 75 8
Glucose Isom. 1 125 64 Lysozyme 1 9 6 Fab
D 0 8 2
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A difficult case Fab-D
Hair has been incorporated within the crystal
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Thanks to
Frederic Villard Daniel Mareque Terese Bergfors
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Workshop
Lysozyme 30 mg/ml 500ul reservoir solution 1
crystal drop each for seeding
Streak seeding
Edge seeding
Solution Microseeding
Heterogeneous seeding